1. Field of the Invention
The present invention concerns a reagent and a method for introducing nucleic acid into animal cells.
2. Background Information
There are currently four major reagents or methods used to introduce DNA into animal cells. These are (1) CaPO.sub.4 -DNA precipitates, (2) DEAE dextran-DNA complexes, (3) electroporation and (4) "LIPOFECTIN".TM. reagent, a transfection reagent marketed by BRL (Life Technologies, Inc., Gaithersburg, Md).
Recently, a liposome-mediated transfection protocol ("LIPOFECTION".TM.) has been reported for the introduction of DNA into animal cells (Philip L. Felgner, Thomas R. Gadek, Marilyn Holm, Richard Roman, Hardy W. Chan, Michael Wenz, Jeffrey P. Northrop, Gordon M. Ringold and Mark Danielsen, "Lipofection: A Highly Efficient, Lipid-Mediated DNA-Transfection Procedure," Proc. Natl. Acad. Sci. USA, 84, 7413-7417 (1987)). This protocol uses the synthetic cationic lipid DOTMA (N-[1-(2,3-dioleyloxy)propyl] -N,N,N-trimethylammonium chloride). Liposomes composed of DOTMA and a neutral lipid PtdEtn (dioleoylphosphatidylethanolamine) form stable complexes with DNA, and deliver DNA into several eukaryotic cells with higher efficiency and reproducibility than other methods.
A transient expression system that requires transfected DNA to be present in the cytoplasm has recently been described by T.R. Fuerst, E.G. Niles, W. Studier and B. Moss, Proc. Natl. Acad. Sci. USA, 83, 8122-8126 (1986). This system is based on use of a recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. The plasmid DNA containing the gene of interest under control of the T7 promoter is transfected as a CaPO.sub.4 precipitate into the cytoplasm of the vaccinia infected cells where it is transcribed efficiently by the T7 RNA polymerase. The mRNA derived from the transfected gene can be as much as 10% of the total cytoplasmic RNA. This system has facilitated studies of viral glycoprotein translocation (A.S. Shaw, P.J.M. Rottier and J.K. Rose, Proc. Natl. Acad. Sci. USA, 85, 7592-7596, (1988)) and virus assembly (M. Whitt, L. Chong and J.K. Rose, J. Virol., 63, 3569-3578 (1989)), and has allowed for the definition of interacting domains of the lymphocyte glycoprotein CD4 and an intracellular tyrosine protein kinase (A. Shaw, K. Amrein, C. Hammond, D.F. Stern, B.M. Sefton and J.K. Rose, Cell, 59, 627-636 (1989)). A major difficulty with this system was the lack of reproducibility of the transfection step using CaPO.sub.4 precipitates of DNA (Fuerst et al, Proc. Natl. Acad. Sci. USA, 83, 8122-8126, (1986) and F.L. Graham and A.J. Van Der Eb, Virology, 52, 456-467, (1973)). The percentage of cells expressing protein showed a high degree of day-to-day variability, some plasmid DNA preparations were inactive for unknown reasons, and it was not possible to use impure plasmid DNA preparations containing large amounts of RNA (DNA from minipreps).
The variability in the transfection was overcome by using lipofection instead of the CaPO.sub.4 procedure (M. Whitt et al, J. Virol., 63, 3569-3578, (1989)) and a threefold increase over the best expression levels obtained with CaPO.sub.4 was found.
A major drawback to the DOTMA transfection procedure is that the compound itself is not commercially available, and the preformed liposomes containing DOTMA ("LIPOFECTIN".TM. reagent, Life Technologies, Inc., Gaithersburg, Md.) are too expensive for large scale use in transient assays. The cost of lipofection is prohibitive ($145/ml or about $10 per transfection) to laboratories, especially to laboratories which perform thousands of transfections per year.
P.L. Felgner and G.M. Ringold, "Cationic Liposome-Medicated Transfection," Nature, 337, 387-388, (1989) at page 387 report that liposomes comprised of stearylamine or dioctaldecyl-dimethylammonium bromide were inactive in transfection assays.
J.-P. Behr, B. Demeneix, J-P. Loeffler and J.P. Mutul, Proc. Natl. Acad. Sci., USA, 86, 6982-6986, (1989) described a transfection procedure using compacted lipopolyamine-coated plasmids.
It would be advantageous to have a reagent and method for introducing nucleic acids into animal cells using readily available and relatively inexpensive compounds.